Preclinical studies in mice have shown that donor cells can engraft into the central nervous system (CNS) as bone marrow-derived macrophages (BMDM) and functionally replace host microglia. Microglial replacement therapy (MRT) has been used in animal models to provide the CNS with therapeutic proteins, as well as to treat neuroinflammatory and neurodegenerative diseases and metabolic disorders. However, little is known about donor cell engraftment into the CNS in humans because of the difficulties in identifying donor cells in poorly preserved postmortem tissue from patients who have undergone hematopoietic cell transplant (HCT).
In the largest human study characterizing BMDMs in post-transplant patients, Keith Loeb, MD, PhD, of Fred Hutch Cancer Center in Seattle, and colleagues developed a sensitive assay that combines XY fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) to detect, quantify, and characterize BMDMs in the cerebral cortex. The results of the study were published in Blood Advances.
Because the XY FISH assay is not compatible with standard IHC assays, characterizing donor cells in humans is problematic. Nevertheless, “developing a tyramide-based reagent for IHC, which forms a covalently linked fluorochrome that is stable to the proteolytic treatment required to unmask DNA for FISH detection, allowed [us] to combine the two techniques,” Dr. Loeb said.
Employing this assay, researchers studied frontal cortex biopsies from 19 female sex-mismatched patients who had undergone HCT, with the aim of detecting and enumerating male donor-derived cells. They found that pretreatment conditioning, post-transplant survival, and multiple HCTs can increase donor cell engraftment.
The donor cell percentage was defined as the number of cells with a Y chromosome divided by the number of FISH-positive cells. On average, microglial cells represented 12% of cellularity within the cortical gray matter; donor cell percentage was calculated relative to that.
Researchers confirmed preclinical studies showing the effect of pretransplant conditioning on engraftment: after myeloablative conditioning, male donor cells comprised on average six-fold more of the microglial population than non-myeloablative cases (8.1% vs. 1.3%). Additionally, researchers observed that in Busulfan-based myeloablative conditioning regimens, donor cells comprised an average of 6.8% of microglia, indicating this regimen is as effective as other myeloablative treatments in supporting bone marrow macrophage engraftment while “reducing the pathogenicity and morbidity of the procedure,” Dr. Loeb said.
Researchers also found a higher average of donor cell engraftment in cases with multiple transplants than in those with a single transplant (16.3% vs. 6.35% of microglial cells).
Moreover, they noted a correlation between donor cell number and time after transplant: 25% of the microglial population in the longest surviving patient (15.56 years) were BMDMs, and donor cells in multiply transplanted cases fit a linear regression model (R2=0.99), suggesting an increase in donor cell number by proliferation or ongoing engraftment. Still, it is unclear if this higher number of donor cells is the result of self-replicating engrafted BMDM or repeat HCTs. “Larger studies are required to better define how the donor cell population in the brain expands in long-term survivors,” researchers said.
Researchers cited the small sample size and limited number of longer surviving patients as limitations to their study.
In the current study, engraftment rates were as high as 25%. The success of engraftment “sets the stage for future research on MRT as a therapeutic option for disorders of the CNS,” researchers noted. Dr. Loeb cited “two recent animal studies that achieved about 90% donor cell replacement of microglia in the brain using a combination of immune ablation and conditioning.” In a murine model of MRT, a combination of pretransplant microglial depletion via CSF1R inhibition and conditioning yielded more than 92% donor cell engraftment, while in another model, post-transplant CSF1R inhibition resulted in near complete BMDM engraftment.
“The future of MRT is bright with new techniques designed to decrease problems associated with pretransplant conditioning and to dramatically increase donor cell engraftment in the CNS,” Dr. Loeb said. However, to treat patients before irreversible tissue and neuronal damage occurs, “we need better ways of detecting these disorders early on and then minimizing the potential morbidities of the transplant or cell therapy procedure.”
Any conflict of interest declared by the authors can be found in the original article.
Reference
Loeb K, Loeb A, Pattwell S, et al. Donor bone marrow derived macrophage engraftment into the central nervous system of allogeneic transplant patients [published online ahead of print, 2023 June 14]. Blood Adv. doi: 10.1182/bloodadvances.2023010409.
